lecture 514.094 the pathophysiology of allergy chapter · pdf filelecture 514.094 of the...

Lecture 514.094 of the Medical University Vienna

The Pathophysiology of AllergyChapter 2: Type I allergy: Mechanisms in

the effector phase and AnaphylaxisEva Untersmayr-Elsenhuber

Department of Pathophysiology and Allergy Research

Lecture 514.094 The Pathophysiology of Allergy

Chapter 2: Type I allergy: Mechanisms in effector phaseand Anaphylaxis

Eva Untersmayr-ElsenhuberDepartment of Pathophysiologyand Allergy ResearchMedical University Vienna





Topics of lecture• Clincal presentation of allergic reactions

• Key players in allergic inflammation

• Mechanisms of anaphylactic reactions

− IgE− Allergen− Mast cells− Eosinophils, inflammatory cells





Clinical presentation of allergic reactions

http://www.raksar.com/shop/r/raksar/img-lib/con_20051229134550_i.JPG

Allergy against inhalative proteins

• Respiratory symptoms

• Cutaneous and gastrointestinal symptoms

Wheezing, rhinitisConjunctivitisAsthma

EczemaOral allergy syndrom

Mild à severeIn all age groups

Gibson PG et al. Lancet 2010

Katelaris CH. Curr Opin Allergy Clin Immunol. 2010






http://sip.uki.at/img/bauchschmerz/Schmerz_Bauchschmerz.jpg

Allergy against food compounds

• Gastrointestinal symptoms

• Respiratory and cutaneoussymptoms

• Generalized anaphylactic symptoms

Abdomial painNausea, vomitingDiarrhea

Rhinitis, conjunctivitisAsthmaEczema, urticaria, angioedema

Sicherer SH, Sampson HA. JACI 2010






http://www.allergiewelle.de/wp-content/uploads/2009/11/angioodem-fdgarrett.jpg

Allergy against drugs

• Respiratory symptoms

• Cutaneous symptoms

• Generalized anaphylactic symptoms

Wheezing, rhinitisConjunctivitisAsthma

EczemaUrticariaAngioedema





Time course of allergic reactionsimmediate Late phase

immediate Late phase

Immediate reactionOnset within secondsdue to preformed or rapidly synthesized mediators à vascular permeabilityà contraction of smooth muscle

Late phase reactionInduced synthesis and release of mediators à recruiting of eosinophilsand Th2 lymphocytesà second phase of smooth muscle contraction, sustained edema, airway hyperreactivity

www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=imm&part=A1734





Time course of allergic reactions

Key players in immediate reactionsIgE bound via FcεRI to mast cells and basophilsà mediator release



Laché M et al. Nat Rev Immunol 2006





Time course of allergic reactions

Key players in late phasereactionNetwork of inflammatory cells:eosinophils (50% of infiltrate), allergen-specific T-cells, mast cells, basophils, Th1 cells



Laché M et al. Nat Rev Immunol 2006





Chronic allergic inflammation

Galli SJ et al. Nature 2008

Due to repetitive or persistantallergen exposureInnate immune cells (eosinophils, basophils, neutrophils and monocyte/macrophages) and adaptive immune cells (Th2-cells, other T-cells, B-cells) take up residence in tissue





Mean serum concentration:IgE: 0.02-0.5 mg/mlIgG: 8-16 mg/ml

Percentage of total Ig:IgE: 0.002 %IgG: 80 %

Serum half life time: 2 days

Peak IgE levels occure 4-6 weeks after peak of pollenseason

Total IgE >1000ng/mLàmajor diagnostic criteria forallergic bronchopulmonaryaspergillosis

IgE antibodiesIgE IgG





Diseases with elevated IgE levels

• Atopic diseases• Parasitic infections (eg.

Strongyloidiasis, ascariasis, schistosomiasis)

• Nonparasitic infections (eg. EBV, CMV, HIV, M. tuberculosis)

• Inflammatory disease (eg. Kimura disease, Churg-Stauss vasculitis, Kawasaki disease)

• Malignancies (eg. Hodgkin lymphoma, IgE myeloma)

• Cutaneous diseases (eg. Bullouspemphigoid)

• Cystic fibrosis• Nephrotic syndrome• Primary immunodeficiency

diseases (eg. Hyper-IgEsyndrome, Wiskott-Aldrich syndrome, Omenn syndrome, immune dysregulation, X-linkedinheritance, atypical DiGeorgesyndrome





Ig switch: - transcription through upstream constant switch region- DNA cleavage of ssDNA at transcription site- DNA repair: recombine VDJ domain + new C domain

Immunoglobulin class switch

IgM expressing B-cell IgG1 expressing B-cell

2 signals for IgE switch1) IL4 or IL13 via STAT6:

activates transcriptionat Sε

2) CD40L (T-cells) andCD40 (B-cells):activates DNA switchrecombination





Sensitisation to allergens

Lymph nodesor mucosa


Airway Basophils, mastcells, NK T cells,…

Dendritic cell samplingEntrance through disrupted epitheliaProtease activityà cleaving of epithelialtight junctions





High affinity IgE receptor FcεRITetrameric FcεRIαβγ2

According to: Kraft S, Kinet JP. Nature Immunol 2007

αγ2 β

mast cells, basophils

αβ γ2

Antigen independent

effects

Increased cell survival

Antigen dependenteffects

Mediator release





Trimeric FcεRIαγ2

According to: Kraft S, Kinet JP. Nature Immunol 2007

α

γ2

MHC class II

monocytes, macrophages, dendritic cells, Langerhans cells, eosinophils

Mediator release





à ligand:receptor = 1:1à high affinity (1010 M-1) due to low dissociation rate, each binding site has lower intrinsic affinity

1 FcεRIα chain with 2 asymmetric IgE interaction sites:

- 4 solvent-exposed tryptophans àlarge hydrophobic surface- C-C’ loop in the receptor D2 domain

bind dimeric, symmetric IgE Fc (Cε3)

FcεRI and IgE interaction

Garman SC et al. Nature 2000Wan T et al. Nature Immunol 2002

Metzger H. Immunol Rev 1992

Gould HJ et al. Nat Immunol Rev 2008





Glycoprotein, 45 kDa,single chainhomology to C-type(Ca2+-dependent) lectinsà IgE and CD21 bind tolectin domain

lower affinity for IgE (107 M-1)

B-cells, activated T-cells, monocytes, macrophages, eosinophils, Langerhans cells, intestinal epithelial cells, platelets

Low affinity IgE receptor FcεRII/CD23

Mossalayi MD et al. EMBO J 1992





CD23 regulates IgE synthesis

Down-regulation of IgE synthesis

Co-crosslinking of mCD23 and mIgE by allergen-IgEcomplexà Inhibition of B-cell proliferation and IgE production

à Induction of B-cell apoptosis à B-cell populationregulation

B-cell surface

Luo HY et al. J Immunol 1991Sherr E et al. J Immunol 1989

Hibbert RG et al. JEM 2005

From Hibbert RG et al. JEM 2005

Up-regulation of IgE synthesisCo-crosslinking ofmIgE and CD21 bytrimeric sCD23

Aubry JP et al. Nature 1992Hibbert RG et al. JEM 2005

IgE binding tomCD23 protectsmCD23 againstproteolysis andprevents formationof sCD23





Glycoproteinslow dose à activation of IL4-producing CD4-T-cells

low molecular weight (10-70 kDa) à diffusion through epithelia facilitatedsolubility

Stability stable to head, acid, proteasesenzymatically active, protease some are enzyme inhibitors

abundance in food

Characteristics of allergens

Pollen allergens Food allergens

http://www.umass.edu/tei/TEI/images/UMassEnv/pollen.gif

http://blog.friendseat.com/wp-content/uploads/2010/09/Allergy-Foods.jpg





high binding affinity and slow dissociation rates of IgE to the allergenIgE: 10-10 bis 10-11 MIgG: 10-7 bis 10-8 M

IgE – allergen interaction

Kim KE et al. Mol Immunol 1996Pierson L et al. J Immunol Meth 1998Jackola DR et al. Mol Immunol 2002Hantusch B et al. Immunol Lett 2005

à IgE affinity to allergen among the highest biologicallyrelevant binding strength

IgE epitopes on allergens are mainly conformational

IgE to conformational rather than linear epitopescorrelate with tolerance development in milk and egg allergic children

Foote J et al. PNAS USA 1995

Xia L et al. Mol Immunol 2010Padavattan S et al. J Immunol 2009

Pedraza-Escalona M et al. Mol Immunol 2009

Järvinen KM et al. Allergy 2007Vila L et al. Clin Exp Allergy 2001

repeated epitope presentation on allergen surface required for crosslinkingà allergens are multimers Tan YW et al. J Biol Chem 2009

Schöll I et al. J Immunol 2005





Morphology of mast cell

http://www.bu.edu/histology/i/22602ooa.jpg

Tissue based inflammatory cellsRespond to signals of innate and adaptive immunity with immediate and delayed release ofinflammatory mediators

20 μm diameterOvoid or irregularly elongated cells with ovoid nucleusAbundant metachromatic cytoplasmatic granules (metachromatic stainingdue to sulfated proteoglycans)

cKIT (CD117) and FcεRI positiveOther cell-surface receptors depending on location and activation:FcγRIIa (in resting state) FcγRI (CD64) (in presence of IFN-γ)β2-adrenergic receptor, adenosine receptor A2B, prostaglandin E2 receptor,C3a, C5a-receptor, IL-3R, IL-4R, IL-5R, IL-9R, IL-10R, GM-CSFR, IFN- γR,CCR3, CCR5, CXCR2, CXCR4, nerve growth factor R, TLRs, …





Mast cell maturation and tissue distribution

Hematop. Stem cell

Mast cellprogenitor

cKIT (CD 117) – SCF Ligand

Arise from CD34+ progenitors


Mucosalmast cell

Connectivetissuemast cell

Tryptase pos. In respiratoryand GI mucosaàincreasedwithinflammation

Tryptase, mast cell-specific chymase pos. In connective tissue (dermis, submucosa of GI tract, heart, conjunctivae, perivascular)Small bowel of end-stage immunodeficiencies

Phenotype of maturemast cells depends on growth factor milieu





• Antigen-crosslinked surface-bound IgE

• Divalent Ab against IgE Fc region

• Anti-idiotypic Ab

• Anti- Fc receptor Ab

• Covalent cross-linked IgE

• Lectins

• Complement (C3a, C5a) throughC3aR, C5aR (CD88)

• Nerve growth factor

• IgG by FcγRI

• TLR ligands (eg. TLR3 dsDNAà IFN-γ production in mast cells)

Mechanisms of mast cell activation

C3aC5a7





Mast cell mediators

Granule-ass. preformed mediators• Histamine• Neutral proteases (tryptase in MCT,

tryptase, chymase, cathepsin G, carboxypeptidase in MCTC)

• Proteoglycans (heparin, chondroitinsulfates) neg. chargedà complexeswith histamine

• Chemotactic and activating factors(ECF-A, NCF)

Newly formed mediators• Lipoxygenase pathway products:

SRS-A (LTC4, LTD4), leukotrienes(LTB4)

• Cyclo-oxygenase products: prostaglandines and thromboxanes

• PAF





Effector function of mediatorsChemo-attractants• NCF• ECF-A• LTB4

Activators• Histamine• PAF• Tryptase• Kininogenase

Spasmogens• Histamine• PGD2• LTC4• LTD4

preformed newly formed

Attractants ofNeutrophils, eosinohils, monocytes, basophils

Vasodil., Vascular permeability

Kininsà Vasodil. à edemaProteolyt. enzyme, activates C3Microthrombi

• Bronchial smooth muscle contraction

• Mucosal edema• Mucus secretion





Clinical aspects of mast cell activation

Allergen dosageand entranceroute are decisivefor clinical reaction

MCTMCTCintravenoushigh dose

subcutaneouslow dose

inhalationlow dose

uptake withfood

Mast cell activation

Anaphylaxis Wheal andflare reaction

Allerg. rhinitisAsthma

Nausea, pain, vomiting diarreha





Morphology of basophils

http://microanatomy.net/blood/basophil3.jpg

Share many features with mast cells: FcεRIsecretion of Th2 cytokinesmetachromic stainingrelease of hisatmine after activation

But distinct lineage

5-8 μm diameter, segmented condensed nucleus, little proliferative capacityRapid and potent expression of IL-4 and IL-13Express Cytokine receptors (IL-3R, IL-5R, GM-CSF receptor)

Chemokine receptors (CD11c, CD11c, CD35 and CD88)Ig receptors (FcεRI, FcγRIIb)TLR





http://focosi.altervista.org/blood-cell-development.jpg

Basophil development and activation• CD34+ progenitors• Differentiate and mature in bone marrow• Circulate in periphery, <1% peripheral

leukocytes• Differentiation driven by IL3• Express integrins and chemokine

receptorsà able to inflitrateinflammed tissue (skin in AD, airway of respiratory allergies)

Activation via IgE crosslinkingC3a, C5a, TLR2 and TLR4 à IL-4, IL-13 secretionand potentiation of IgE activationIL-33 through ST2 receptor





Mediators: preformed: Histamine, less heparin, low tryptase levelsnewly synthesized: LTC4, LTD4, LTE4, no PGD2 productioncytokines: IL-4, IL-13, GM-CSF à source of early IL-4 for Th2 cell differentiation and amplification of IgE synthesis

Role in health and disease

Karasuyama H et al. Nat Rev Immunol 2009Min B. Nat Immunol 2008

Sullivan BM et al. Immunity 2009

Sullivan BM et al. Immunity 2009

• Physiological function remains unknown (host defense against parasites?)• Innate immunity (TLR2 expression)• Predominant source of IL-4 in allergen and helminth parasite activated

PBMCs• In late-phase allergic responses, found in increased numbers in lungs of

asthma patients dying of asthma





Features of mast cells and basophils

OriginMaturationLifespanLocationSizeNucleusGranulesPeptido-glycansTryptasecontent

Mast cells Basophils

Hematopoietic stem cellConnective tissueMonthsTissue6-12 μmOval or roundSmaller and moreHeparin and chondroitinsulfatesHigh

Hematopoietic stem cellBone marrowDaysIntravascular circulation5-8 μmSegmentedLarger and fewerPredom. chondroitinsulfatesLow

Stone KD et al. JACI 2010





Morphology of eosinphils

http://student.nu.ac.th/wuth_web/eosinophil2.jpeg

Blood and tissue eosinophilia hallmarks for• helminth infection• allergy and asthma, • eosinophilic gastrointestinal disorders• other rare disorders (eg. Hypereosinophilia Churg-

Strauss syndrom)

Bilobed nucleus, highly condensed chromatinStained with acidic dye eosin

2 major types of granules:Specific granules with electron-dense core, contain cationic proteins:

major basic protein MBP, eosinophil peroxidase EPO, eosinohil cationic protein ECP, eosinophil-derivedneurotoxin EDN

Primary ganules similar to those found in other granulocytesenriched in Charcot-Lyden crystal protein





Cell surface receptors

http://www.scq.ubc.ca/wp-content/uploads/2006/07/cellsurfaceR.gif

• IgG receptor (FcγRII/CD32)• IgA receptor (FcαRI/CD89)• Complement receptors (CR1, CR3,

CD88 for C3a and C5a)• Cytokine receptors (IL-3R, IL-5R;

receptors for GM-CSF, IL-1α, IL-2, IL-4, IFN-γ, TNF-α)

• Chemokine receptors (CCR1, CCR3)• Adhesion molecules (very late antigen4, α2β7, siglec-8)• Leukotrien receptors (CysLT1R, CysLT2R, LTB4 receptor)• PG receptors (PGD2 type 2 receptor)• PAF receptor• TLR7/8• FcεRI expression is minimal, does not activate eosinophils, unclear

functional significance





Development and activation

http://www.tpub.com/content/medical/14295/img/14295_284_1.jpg

IL-5, IL-3, GM-CSF à eosinophil developmentfrom CD34+ progenitor cells

Release from bone marrow into circulation after IL-5 and CCL11 (eotaxin-1, via CCR3) stimulation

IL-4, IL-13 promote eosinophil trafficking tomucosal tissue by eotaxin (CCL11, CCL26) upregulation

Eosinophil chemotactic factors: PAF, LTD2, C5a, CCL5 (RANTES)





http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=imm&part=A1233&rendertype=figure&id=A1239

Effector function of eosinophilsRelease of proinflammatory mediators:Granule-stored cationic proteins, newly synthesizedeicosanoids and cytokines

MBP 50% of granule protein massacitivity against parasites (helminths, schistosomula)in asthma: serum and BALF MBP correlates withbronchial hyperresponsiveness

EDN, ECP RNAse activitytoxicity to parasites and ssRNA viruses

EPO 25% of granule protein masstoxic to microorganisms and host cells

Role of eosinophils in airway remodelling, airway hyperreactivity, and mucusproduction





The allergic effector unit

http://personalpages.manchester.ac.uk/staff/j.gough/lectures/TIB/3imm_intro/mast2.jpg

http://pathmicro.med.sc.edu/bowers/neut-em.jpg

Soluble mediatorsmodulate the reciprocalinteraction between mastcells and eosinophils

Mast cell Eosinophil

GM-CSF, IL-3, IL-5, TNF-α, Heparin, LTC4, LTD4, LTE4, Chymase, PGD2, LTB4, IL-2, Tryptase, Histamine, PAF

SCF, NGF, MBP, Eotaxin, EDN, LTC4, LTD4, LTE4, ECP, EPO

Minai-Fleminger Y, Levi-Schaffer F. Inflamm Res 2009





The allergic effector unit

http://personalpages.manchester.ac.uk/staff/j.gough/lectures/TIB/3imm_intro/mast2.jpg

http://pathmicro.med.sc.edu/bowers/neut-em.jpg

Physical interactions between mast cells and eosinophils

Mast cell Eosinophil

Receptor-ligand couples

TRAIL – TRAIL ligandCD300a – CD300a

CD48 – CD244ICAM-1 – LFA-1

Minai-Fleminger Y, Levi-Schaffer F. Inflamm Res 2009





Early phase of allergy


Allergen-specific IgEcross-linkingàFcεRI aggregation àmast cell activation

Mediator release

BronchoconstrictionVasodilatationInceased vascularpermeability, increased mucusproduction

Transition to latephase: leukocyte recruitment





Late phase of allergy


Action of innate andadaptive immune cells and secretion ofinflammatorymediators fromtissue-resident cells

Degradation of type III collagen, injury ofepithelial cellsBronchoconstricition





Chronic allergic inflammation


Prolonged allergen exposure

• Increased mucusproduction, thickening ofsubepithelial tissue

• Formation of EMTU (epithelial-mesenchymaltropic unit)

• Smooth musclehyperplasia

• Severe narrowingof airway lumen





Tissue remodelling in asthma

Galli SJ et al. Nature 2008Normal small bronchus Severe asthmaMucus fills airway lumenNumerous goblet cellsThickened lamina reticularisMany eos and MC in submucosaMore bronchial smooth muscle





Anaphylaxis= Serious allergic reaction with rapid onset, which can cause death

Anaphylaxis is unpredictable, can occur in anyone, anywhere at any timeUnderrecognized by patients and underdiagnosed by MDs

Incidence: doubled from 21/100.000 person/year (1980s) to 50/100.000 person/year (1990s)

with 70/100.000 person/year younger than19 years

Triggers: Food (peanut, treenut, shellfish, fish, milk, eggs)Medication (β-lactam antibiotics)Insect stings/bitesNatural latex rubberIgG-Ag complexesComplement and coagulation system activation

exercise, cold air or water, medication Simons FER. JACI 2009





http://www.bio.davidson.edu/courses/immunology/Students/spring2006/Witcher/figure%2012-11.jpg

Mechanisms





Patient-specific risk factorsSevere concomitant allergic rhinitis and eczemaSevere asthmaChronic obstructive pulmonary diseaseOther respiratory diseasesCardiovascular diseasesMastocytosis and clonal mast cell disordersDiseases impeding prompt recognition (CNS, psychiatric disorders,..)

Increased risk of severe, life-threatening or fatal anaphylaxis

Diagnosis of anaphylactic event: histamine in serum and 24h urinetryptase

Confirmation of anaphylactic trigger: History, skin tests, allergen-specific IgEChallenge tests

Simons FER. JACI 2009





Therapy of anaphylaxisPreventive measures:• Avoidance of specific triggers• Immunomodulation (oral desensitization, sc. anti-IgE, TCM medication,

immunotherapy with insect venom)

Emergency preparedness:• Self-injectable epinephrine

Leung DYM et al. N Engl J Med 2003Simons FER. JACI 2006

Srivastava KD et al. JACI 2009

Emergency treatment:• Stop allergen exposure• O2 and volume substitution• Epinephrine gavage• Antihistamines• Corticosteroids• β2-Symptatomimetica





The correct use can save lives!

Patient training for EpiPen use





Thank you foryour attention!

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